Compositions for treating multiple sclerosis

ABSTRACT

Compositions containing Tr1 cells directed to a multiple sclerosis associated antigen and methods for treating multiple sclerosis are presented.

FIELD OF THE INVENTION

The present invention relates to the field of treatment of autoimmunedisease, such as multiple sclerosis. More particularly, it concerns amedicament comprising Tr1 cells directed against multiplesclerosis-associated antigen.

BACKGROUND

Multiple sclerosis is a demyelinating and chronic inflammatory diseaseof the central nervous system. The histopathologic hallmarks of thedisease include focal infiltration of both CD4+ and CD8+ T cellstogether with other inflammatory cells in the white matter anddemyelination with evidence of some axonal damage. The myelin proteinsthought to be the target of an immune response in multiple sclerosisinclude myelin basic protein (MBP), proteolipid protein (PLP), myelinassociated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein(MOG).

A variety of therapeutic approaches are now available in humans to treatmultiple sclerosis. However, no curative treatments exist for multiplesclerosis. While a number of compounds, including corticosterioids andmodified beta interferon, can reduce some symptoms of multiplesclerosis, they have proven to have serious side effects or otherwisebeen shown to be less than desirable for long term use.

One promising treatment for multiple sclerosis is described in WO02/077025 which discloses the use of peptide analogs of myelin basicprotein (MBP). Compositions comprising these analogs are reportedly ableto ameliorate symptoms of MS without excessive side effects. Moreover,use of peptide analogs to myelin constitutive proteins were also shownto be effective in treating the symptoms of experimental allergicencephalomyelitis (EAE), an organ specific immune disorder often used inmice as a model for MS. However, several phase II clinical trials had tobe halted due to the poor tolerance of the altered MBP peptide at thedose tested (Bielekova et al., nature medicine, 2000, (6) 10: 1167 etKappos et al., nature medicine, 2000, (6) 10: 1176).

Another promising treatment for multiple sclerosis is also described inEP0587735. Said treatment is based on the rational that immunologicexposure to a peptide closely resembling an autoreactive TCR fragmentshould enhance Th2 cells priming/recognition, and thus help to maintaincytokine regulatory control over Th1-mediated inflammation. Clinicaltrials have demonstrated acceptable safety and tolerability of thistreatment by the patients; however, this treatment is only effective on50% of the immunized patients. US2004/0087018 describes a method fortreating multiple sclerosis in a patient in need thereof, comprisingadministering antigen-specific IL-10 producing cells to said patienttogether with the soluble antigen, preferably simultaneously.

The inventors surprisingly found that the administration of Tr1 cellsdirected against a multiple sclerosis-associated antigen, withoutco-administration of the soluble antigen, dramatically inhibits thedevelopment of EAE in immunized mice.

Therefore, the Applicant aim to provide another type of treatment formultiple sclerosis based on the use of Tr1 cells.

SUMMARY OF THE INVENTION

The present invention is directed to a composition comprising at leastone Tr1 cell population directed against a multiple sclerosis-associatedantigen. Said multiple sclerosis-associated antigen is preferablyselected from the group comprising myelin basic protein, myelinassociated glycoprotein, myelin oligodendrocyte protein, proteolipidprotein, oligodendrocyte myelin oligoprotein, myelin associatedoligodendrocyte basic protein, oligodendrocyte specific protein, heatshock proteins, oligodendrocyte specific proteins NOGO A, glycoproteinPo, peripheral myelin protein 22, 2′3′-cyclic nucleotide3′-phosphodiesterase.

Another object of the present invention is to provide a medicament or apharmaceutical composition comprising the composition of the invention.

The present invention relates also to a method for treating multiplesclerosis in a subject in need thereof, comprising administering to saidsubject an effective amount of the medicament or the pharmaceuticalcomposition of the invention. In a preferred embodiment, the medicamentor the pharmaceutical composition to be administered to a subject inneed thereof comprises Tr1 cells autologous to the cells of saidsubject.

In another embodiment of the present invention, the method for treatingmultiple sclerosis in a subject in need thereof comprises theadministration to said subject of an effective amount of the medicamentor the pharmaceutical composition of the invention in combination withanother therapeutic agent used for treating multiple sclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Cytokine secretion profile of differentiated cells.

Naïve CD4+ cells submitted to differentiation regimens were activatedwith anti-CD3+ anti-CD28 monoclonal antibodies during 48 hours. Culturesupernatants were then tested by ELISA for the presence of IL-4, IL-10and IFN-gamma.

FIG. 2: Effect of anti-MOG₃₅₋₅₅CD4+ T cells administration in EAE pronemice.

DETAILED DESCRIPTION OF THE INVENTION Definition

The term “Tr cells” as used herein refers to cells having the followingphenotype at rest CD4+CD25-FoxP3- and capable of secreting high levelsof IL-10 and low to moderate levels of TGF-β upon E activation. Tr1cells are characterized, in part, by their unique cytokine profile: theyproduce high levels of IL-10, significant levels of TGF-β andintermediate levels of IFN-γ, but little or no IL-4 or IL-2. Thecytokine production is typically evaluated in cultures of cells afteractivation with polyclonal activators of T lymphocytes such as anti-CD3+anti-CD28 antibodies or Interleukin-2, PMA+ionomycin, Alternatively, thecytokine production is evaluated in cultures of cells after activationwith the specific T-cell antigen presented by antigen presenting cells.High levels of IL-10 correspond to at least about 500 pg/ml, typicallygreater than about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 thousandpg/ml or more. Significant levels of TGF-β correspond to at least about100 pg/ml, typically greater than about 200, 300, 400, 600, 800, or 1000pg/ml or more. Intermediate levels of IFN-γ correspond to concentrationscomprised between 0 pg/ml and at least 400 pg/ml, typically greater thanabout 600, 800, 1000, 1200, 1400, 1600, 1800, or 2000 pg/ml or more.Little or no IL-4 or IL-2 corresponds to less than about 500 pg/ml,preferably less than about 250, 100, 75, or 50 pg/ml, or less.

The term “antigen” as used herein refers to a protein, or peptide,associated with a particular disease for which the cells of thisinvention are being used to modulate, or for use in any of the methodsof this invention. In one embodiment, the term “antigen” may refer to asynthetically derived molecule, or a naturally derived molecule, whichshares sequence homology with an antigen of interest, or structuralhomology with an antigen of interest, or a combination thereof. In oneembodiment, the antigen may be a mimetope. A “fragment” of the antigenrefers to any subset of the antigen, as a shorter peptide. A “variant”of the antigen refers to a molecule substantially similar to either theentire antigen or a fragment thereof. Variant antigens may beconveniently prepared by direct chemical synthesis of the variantpeptide, using methods well-known in the art.

The term “subject” as used herein refers to a mammal, in particular ahuman being.

The term “effective amount” as used herein refers to an amountsufficient to cause a beneficial or desired clinical result (e.g.improvement in clinical condition).

The term “clone” or “clone population” as used herein refers to apopulation of differentiated cells being derived from a uniquedifferentiated cell.

The term “treatment” or “treating” as used herein generally refers to aclinical intervention in an attempt to alter the natural course of theindividual being treated, and may be performed during the course ofclinical pathology. Desirable effects include, but are not limited to,alleviating symptoms, suppressing, diminishing or inhibiting any director indirect pathological consequences of the disease, lowering the rateof disease progression, ameliorating or palliating the disease state,and causing remission or improved prognosis.

The term “autoimmune disease” as used herein refers to an immuneresponse directed against a self-antigen.

Patients having multiple sclerosis may be identified by criteriaestablishing a diagnosis of clinically definite multiple sclerosis.Briefly, an individual with clinically definite multiple sclerosis hashad two attacks and clinical evidence of either two lesions or clinicalevidence of one lesion and paraclinical evidence of another separatelesion. Definite multiple sclerosis may also be diagnosed by evidence oftwo attacks and oligoclonal bands of IgG in cerebrospinal fluid or bycombination of an attack, clinical evidence of two lesions andoligoclonal band of IgG in cerebrospinal fluid. The McDonald criteriacan also be used to diagnose multiple sclerosis. The McDonald criteriainclude the use of MRI evidence of CNS impairment over time to be usedin diagnosis of multiple sclerosis, in the absence of multiple clinicalattacks. Effective treatment of multiple sclerosis may be evaluated inseveral different ways. The following parameters can be used to gaugeeffectiveness of treatment. Two exemplary criteria include: EDSS(extended disability status scale), and appearance of exacerbations onMRI (magnetic resonance imaging). The EDSS is a means to grade clinicalimpairment due to multiple sclerosis. Eight functional systems areevaluated for the type and severity of neurologic impairment. Briefly,prior to treatment, patients are evaluated for impairment in thefollowing systems: pyramidal, cerebella, brainstem, sensory, bowel andbladder, visual, cerebral, and other. Follow-ups are conducted atdefined intervals. The scale ranges from 0 (normal) to 10 (death due tomultiple sclerosis). A decrease of one full step indicates an effectivetreatment.

Exacerbations are defined as the appearance of a new symptom that isattributable to multiple sclerosis and accompanied by an appropriate newneurologic abnormality. In addition, the exacerbation must last at least24 hours and be preceded by stability or improvement for at least 30days. Briefly, patients are given a standard neurological examination byclinicians. Exacerbations are either mild, moderate, or severe accordingto changes in a Neurological Rating Scale. An annual exacerbation rateand proportion of exacerbation-free patients are determined.

Therapy can be deemed to be effective if there is a statisticallysignificant difference in the rate or proportion of exacerbation-free orrelapse-free patients between the treated group and the placebo groupfor either of these measurements. In addition, time to firstexacerbation and exacerbation duration and severity may also bemeasured. A measure of effectiveness as therapy in this regard is astatistically significant difference in the time to first exacerbationor duration and severity in the treated group compared to control group.An exacerbation-free or relapse-free period of greater than one year, 18months, or 20 months is particularly noteworthy.

Clinical measurements include the relapse rate in one and two-yearintervals, and a change in EDSS, including time to progression frombaseline of 1.0 unit on the EDSS that persists for six months. On aKaplan-Meier curve, a delay in sustained progression of disability showsefficacy. Other criteria include a change in area and volume of T2images on MRI, and the number and volume of lesions determined bygadolinium enhanced images. MRI can be used to measure active lesionsusing gadolinium-DTPA-enhanced imaging or the location and extent oflesions using T2-weighted techniques. Briefly, baseline MRIs areobtained. The same imaging plane and patient position are used for eachsubsequent study. Positioning and imaging sequences can be chosen tomaximize lesion detection and facilitate lesion tracing. The samepositioning and imaging sequences can be used on subsequent studies. Thepresence, location and extent of multiple sclerosis lesions can bedetermined by radiologists. Areas of lesions can be outlined and summedslice by slice for total lesion area. Three analyses may be done:evidence of new lesions, rate of appearance of active lesions,percentage change in lesion area. Improvement due to therapy can beestablished by a statistically significant improvement in an individualpatient compared to baseline or in a treated group versus a placebogroup.

Each case of multiple sclerosis displays one of several patterns ofpresentation and subsequent course. Most commonly, multiple sclerosisfirst manifests itself as a series of attacks followed by complete orpartial remissions as symptoms mysteriously lessen, only to return laterafter a period of stability. This is called relapsing-remitting (RR)multiple sclerosis.

Primary-progressive (PP) multiple sclerosis is characterized by agradual clinical decline with no distinct remissions, although there maybe temporary plateaus or minor relief from symptoms.

Secondary-progressive (SP) multiple sclerosis begins with arelapsing-remitting course followed by a later primary-progressivecourse. Rarely, patients may have a progressive-relapsing (PR) course inwhich the disease takes a progressive path punctuated by acute attacks.

PP, SP, and PR are sometimes lumped together and called chronicprogressive multiple sclerosis. A few patients experience malignantmultiple sclerosis, defined as a swift and relentless decline resultingin significant disability or even death shortly after disease onset.

The Present Invention

The present invention relates to a composition comprising at least oneTr1 cell population directed against a multiple sclerosis-associatedantigen.

In one embodiment of the invention, Tr1 cells may be obtained by

-   a) isolating a progenitor cell population from a subject,-   b) obtaining a population of dendritic cells by culturing said    progenitor cell population in the presence of IL-10-   c) contacting cells of step b) with a CD4+ T lymphocyte population    isolated from said subject in the presence of a multiple    sclerosis-associated antigen to allow differentiation of CD4+ T    cells directed to said antigen into the Tr1 cell population, and-   d) recovering the Tr1 cell population from the step c).

In step b), IL-10 is present from 50 to 250 U/ml, preferably at 100 U/mlin the culture medium. Said method for obtaining Tr1 cells is describedin Wakkach et al (Immunity 2003 May; 18(5):605-17).

Said method may also be carried out using Dexamethasone and Vitamin D3,or tolerogenised or immature DCs instead of the DCs of step b).

In another embodiment of the present invention, Tr1 cells may beobtained by:

a) culturing a CD4+ T cell population directed to a multiplesclerosis-associated antigen isolated from a subject in a media with anappropriate amount of IFN-α, andb) recovering the Tr1 cell population.

IFN-α is preferably present in the media at 5 ng/ml. In the step a), themedia may further comprise an appropriate amount of IL-10, preferably at100 U/ml.

In step b), the Tr1 cell population is cultured in a media comprisingIL-15 to allow proliferation, IL-15 being preferably at 5 ng/ml in themedia. Said method for obtaining TR1 cells is described in the patentUS6746670.

In still another embodiment of the invention, Tr1 cells may be obtainedby:

a) in vitro activating a CD4+ T cell population in presence of amultiple sclerosis-associated antigen, presented by artificial antigenpresenting cells, andb) recovering an activated CD4+ T cells comprising at least 10% of Tr1cells. Preferably, the artificial antigen presenting cells express a HLAII system molecule and a human LFA-3 molecule and don't express theco-stimulation molecules B7-1, B7-2, B7-H1, CD40, CD23 and ICAM-1.

Said process, for obtaining Tr1 cells is described in the patentapplication WO02/092793.

In still another embodiment of the invention, Tr1 cells may be obtainedby:

a) in vitro activating a CD4+ T cell population in presence of amultiple sclerosis-associated antigen and an appropriate amount ofIL-10; andb) recovering the Tr1 cell population.

Preferably, IL-10 is present in the media at 100 U/ml. Said method isdescribed in Groux et al. (Nature 1997, 389(6652):737-42).

In still another embodiment of the invention, antigen-specific Tr1 cellsmay be obtained by:

a) stimulating a leukocyte population or a peripheral blood mononuclearcell (PB MC) population with a multiple sclerosis-associated antigen,b) recovering the antigen-specific Tr1 cell population from thestimulated population,c) optionally expanding said antigen-specific Tr1 cell population.

Leukocytes encompass several types of cells, which are characterized bytheir importance, their distribution, their number, their lifetime andtheir potentiality. These types are the following: the polynuclear orgranular leukocytes, among which one finds the eosinophilic, theneutrophilic and the basophilic leukocytes, and the mononuclear cells,or peripheral blood mononuclear cells (PBMCs), which are large whiteblood cells and consist in the cell types of the immune system(lymphocytes and monocytes). The leukocytes or the PBMCs can beseparated from the peripheral blood by any method known to those skilledin the art. Advantageously, for the separation of the PBMCs,centrifugation may be used, preferably density gradient centrifugation,preferably discontinuous density gradient centrifugation. An alternativeis the use of specific monoclonal antibodies. In certain embodimentsPBMC are typically isolated from the whole blood product by means ofFicoll-Hypaque, using standard procedures. In other embodiments thePBMCs are recovered by means of leukapheresis.

Said method is described in the patent application WO2007/010406.

In still another embodiment, Tr1 cells may be obtained by:

a) culturing a leukocyte population or a peripheral blood mononuclearcell (PBMC) population with mesenchymal stem cells in the presence of amultiple sclerosis-associated antigen,b) recovering the Tr1 cell population.

Said method can also be carried out with nave or memory T cells insteadof PBMC or leukocytes.

The Tr1 cell population thus obtained may further be expanded by culturein presence of cytokines such as Interleukine-2 and Interleukine-4.Alternatively, Interleukine-15 and Interleukine-13 could also be used inTr1 cell expansion cultures.

In the methods described above, Tr1 cells can be characterized by theidentification method described in WO2005/000344. Said identificationmethod of Tr1 cells is based on the detection of the simultaneouspresence of expression products of genes coding CD4 molecule andmolecules from the group comprising CD18 and/or CD11a, and CD49b. Tr1cells can be identified and/or purified by Elisa, flow cytometry, orimmunoaffinity methods with antibodies directed against said markers.

Tr1 cells can also be enriched by positive selection or negativeselection using flow cytometry or magnetic beads. Such methods are alsodescribed in WO2005/000344.

In another embodiment of the present invention, the Tr1 cells directedto a multiple sclerosis-associated antigen may be expanded by the invitro method described in WO2006/108882. Said method comprises:

a) cultivating at a temperature T1 inferior to 35° C., in a culturemedium Mf, feeder cells such as insect feeder cells, said temperature T1allowing the proliferation of feeder cells and said feeder cellsexpressing factors which interact with the following cell surfaceproteins:

-   -   the CD3/TCR complex,    -   the CD28 protein,    -   the IL-2 receptor,    -   the CD2 protein,    -   the IL-4 receptor,        b) contacting the feeder cells obtained in step a) cleared or        not of their culture medium Mf, with the Tr1 cell population        contained in the culture medium Mp, wherein said culture medium        Mp does not initially contain the factors cited in step a), in        order to obtain a mixture containing the Tr1 cell population,        the feeder cells and the culture medium Mp,        c) cultivating the mixture obtained at step b) at a temperature        T2 which is at least 35° C., said temperature being chosen such        that the Tr1 cell population proliferates and the feeder cells        do not proliferate,        d) recovering the Tr1 cell population such expanded.

Examples of factors which interact with the above mentioned cell surfaceproteins include:

-   -   a modified anti-CD3 antibody, wherein the anti-CD3        intracytoplasmic domain of the CD3 heavy chain is replaced with        a transmembrane domain,    -   the CD80 or CD86 protein,    -   the IL-2 secreted by the feeder cells,    -   the CD58 protein,    -   an interleukin selected from the group comprising IL-4 and        IL-13.

In a preferred embodiment of the present invention, said Tr1 cellsdirected to a multiple sclerosis associated antigen may be cloned byusing conventional methods for cloning T cells.

In preferred embodiment of the present invention, said compositioncomprising at least one Tr1 cell population directed against a multiplesclerosis-associated antigen or at least one clone of Tr1 cell directedagainst a multiple sclerosis-associated antigen may be frozen to bestored.

In a preferred embodiment of the present invention, said multiplesclerosis-associated antigen is selected from the group comprisingmyelin basic protein (MBP), myelin associated glycoprotein (MAG), myelinoligodendrocyte protein (MOG), proteolipid protein (PLP),oligodendrocyte myelin oligoprotein (OMGP), myelin associatedoligodendrocyte basic protein (MOBP), oligodendrocyte specific protein(OSP/Claudin11), heat shock proteins, oligodendrocyte specific proteins(OSP), NOGO A, glycoprotein Po, peripheral myelin protein 22 (PMP22),2′3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), fragments,variants and mixtures thereof.

Preferably, said multiple sclerosis-associated antigen is selected fromthe group comprising myelin basic protein (MBP), proteolipid protein(PLP) and myelin oligodendrocyte protein (MOG) peptides and fragments,variants and mixtures thereof.

More preferably, said multiple sclerosis-associated antigen is selectedfrom the group comprising MBP 82-98, MBP 83-99, MBP 151-170 for HLA-DR2positive subjects.

More preferably, said multiple sclerosis-associated antigen is selectedfrom the group comprising MOG 35-55, MOG 21-40, MOG 41-60, MOG 71-90,MOG 81-100, MOG 111-130, MOG 63-37 for HLA-DR2 positive subjects.

More preferably, said multiple sclerosis-associated antigen is selectedfrom the group comprising MBP 111-129, MBP 116-123 for HLA-DR4 positivesubjects.

More preferably, said multiple sclerosis-associated antigen is selectedfrom the group comprising MOG 21-40, MOG 97-108, MOG 71-90, MOG 181-200for HLA-DR4 positive subjects.

Another object of the present invention is to provide a medicamentcomprising a composition as described here above.

The present invention also intends to provide a pharmaceuticalcomposition comprising a composition as described here above incombination with one or more pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers useful herein are conventional.Remington's Pharmaceutical Sciences 16^(th) edition, Osol, A. Ed. (1980)describes composition and formulations suitable for pharmaceuticaldelivery of the composition of the present invention. In general, thenature of the carrier will depend on the mode of administration beingemployed. For instance, parenteral formulations usually compriseinjectable fluids that include pharmaceutically and physiologicallyacceptable fluids such as water, physiological saline, balanced saltsolutions, aqueous dextrose, sesame oil, glycerol, ethanol, combinationsthereof, or the like, as vehicle. The carrier and composition can besterile, and the formulation suits the mode of administration. Inaddition to biological neutral carriers, pharmaceutical compositions tobe administrated can contain minor amounts of non toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate. The composition can be a liquid solution, suspension,emulsion.

In one embodiment of the invention, said medicament or pharmaceuticalcomposition as described here above consists essentially of at least oneTr1 cell population directed against a multiple sclerosis-associatedantigen.

In another embodiment of the invention, said medicament orpharmaceutical composition as described here above consists essentiallyof at least one clone of a Tr1 cell population directed against amultiple sclerosis-associated antigen.

As used herein, “consists essentially of” refers to a medicament or apharmaceutical composition, wherein at least 70%, preferably 75%, 80%,85% or 90% of the cells present in the medicament or pharmaceuticalcomposition are Tr1 cells directed against a multiplesclerosis-associated antigen,

In another embodiment of the invention, said medicament orpharmaceutical composition as described here above consists of at leastone Tr1 cell population directed against a multiple sclerosis-associatedantigen or at least one clone of a Tr1 cell population directed againsta multiple sclerosis-associated antigen.

The present invention relates to the use of a composition as describedhere above for the preparation of a medicament or a pharmaceuticalcomposition for treating multiple sclerosis.

An object of the present invention is also a method for treatingmultiple sclerosis in a subject in need thereof, comprisingadministering to said subject an effective amount of a medicament asdescribed here above or a pharmaceutical composition as described hereabove.

According to the invention, the pharmaceutical composition or medicamentas described here above is for treating multiple sclerosis.

According to the invention, the pharmaceutical composition or medicamentas described here above is for use in the treatment of multiplesclerosis.

According to the invention, said pharmaceutical composition ormedicament is not used in combination with the soluble multiplesclerosis-associated antigen.

According to the invention, said pharmaceutical composition ormedicament is not administrated to the subject together with or incombination with the soluble multiple sclerosis-associated antigen.

According to the invention, there is no need for a co-treatment with thesoluble multiple sclerosis-associated antigen to which the Tr1 cells aredirected.

The composition may be formulated for parenteral, intramuscular,intravenous, intra-peritoneal, injection, intranasal inhalation, lunginhalation, intradermal, intra-articular, intrathecal, or via thealimentary tract.

Preferably, the medicament or pharmaceutical composition of theinvention may be administrated by intramuscular, intraperitoneal orintravenous injection, or by direct injection into the lymph nodes ofthe patient, preferably by intravenous injection.

The amount of Tr1 cells directed to a multiple sclerosis associatedantigen effective in the treatment of multiple sclerosis will depend onthe nature of the multiple sclerosis, and can be determined by standardclinical techniques. The precise dose to be employed in the formulationwill also depend on the route of administration, and the seriousness ofthe disease or disorder, and should be decided according to the judgmentof the practitioner and each individual's circumstances. Effective dosescan be extrapolated from dose-response curves derived from in vitro oranimal model test systems.

In one embodiment of the present invention, 10⁴/kg to 10⁹/kg cells areadministrated to the subject. Preferably 10⁵/kg to 10⁷/kg cells and morepreferably about 10⁶/kg cells are administrated to the subject.

In one embodiment of the invention, the subject is administrated withthe medicament at the time when flare-up are demonstrated by a declinein the clinical status of the subject or at the time when inflammatorylesions can be visualized for example by MRI within the central nervoussystem.

In one embodiment of the invention, the subject is administrated oncewith the medicament or the pharmaceutical composition of the presentinvention.

In a second embodiment of the invention, the subject is administratedonce a month with the medicament or the pharmaceutical composition ofthe present invention.

In a third embodiment of the invention, the subject is administratedonce a quarter with the medicament or the pharmaceutical composition ofthe present invention.

In a fourth embodiment of the invention, the subject is administratedonce to twice a year with the medicament or the pharmaceuticalcomposition of the present invention.

In another embodiment of the present invention, the medicament orpharmaceutical composition to be administered to a subject in needthereof comprises Tr1 cells autologous to the cells of said subject.

This means that Tr1 cells will be administrated to the subject they comefrom or that precursors used for the production of Tr1 cells come fromthe subject the Tr1 cells will be administrated to.

In another embodiment of the present invention, the method for treatingmultiple sclerosis in a subject in need thereof comprises theadministration to said subject of an effective amount of the medicamentor the pharmaceutical composition of the invention in combination withone or more therapeutic agent used for treating multiple sclerosis.

The present invention relates to the use of the pharmaceuticalcomposition or medicament of the invention, wherein the administrationto said subject of an effective amount of the medicament or thepharmaceutical composition of the invention is in combination with oneor more therapeutic agent used for treating multiple sclerosis.

Examples of therapeutic agents commonly used for treating multiplesclerosis are the following:

-   -   interferons, e.g., human interferon beta-1a (e.g., AVONEX® or        Rebif®) and interferon beta-1b (BETASERON™; human interferon        beta substituted at position 17; Berlex/Chiron);    -   glatiramer acetate (also termed Copolymer 1, Cop-1; COPAXONE™;        Teva Pharmaceutical Industries, Inc.); and derivatives,    -   fumarates, e.g., dimethyl fumarate (e.g., Fumaderm®);    -   Rituxan® (rituximab) or another anti-CD20 antibody, e.g., one        that competes with or binds an overlapping epitope with        rituximab;    -   mitoxantrone (NOVANTRONE®, Lederle);    -   a chemotherapeutic, e.g., clabribine (LEUSTATIN®), azathioprine        (IMURAN®), cyclophosphamide (CYTOXAN®), cyclosporine-A,        methotrexate, 4-aminopyridine, and tizanidine;    -   a corticosteroid, e.g., methylprednisolone (MEDRONE®, Pfizer),        prednisone;    -   an immunoglobulin, e.g., Rituxan® (rituximab); CTLA4 Ig;        alemtuzumab (MabCAMPATH®) or daclizumab (an antibody that binds        CD25);    -   statins;    -   immunoglobulin G intravenous (IgGIV),    -   Nataluzimab (Tysabri) anti-integrin alpha-4 antibody,    -   the oral CC chemokine receptor 1 antagonist BX471 (ZK811752),    -   FTY720 (fingolimod),    -   antibodies or antagonists of human cytokines or growth factors,        for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12,        IL-15, IL-16, IL-17, IL-18, IL-23, EMAP-I1, GM-CSF, FGF, and        PDGF.    -   antibodies to cell surface molecules such as CD2, CD3, CD4, CD8,        CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their        ligands.    -   FK506, rapamycin, mycophenolate mofetil, leflunomide,        non-steroidal anti-inflammatory drugs (NSAIDs), for example,        phosphodiesterase inhibitors, adenosine agonists, antithrombotic        agents, complement inhibitors, adrenergic agents, agents that        interfere with signaling by proinflammatory cytokines as        described herein, IL-Petal converting enzyme inhibitors (e.g.,        Vx740), anti-P7s, PSGL, TACE inhibitors, T-cell signaling        inhibitors such as kinase inhibitors, metal loproteinase        inhibitors, sulfasalazine, azathloprine, 6-mercaptopurines,        angiotensin converting enzyme inhibitors,    -   amantadine, baclofen, papaverine, meclizine, hydroxyzine,        sulfamethoxazole, ciprofloxacin, docusate, pemoline, dantrolene,        desmopressin, dexamethasone, tolterodine, phenytoin, oxybutynin,        bisacodyl, venlafaxine, amitriptyline, methenamine, clonazepam,        isoniazid, vardenafil, nitrofurantoin, psyllium hydrophilic        mucilloid, alprostadil, gabapentin, nortriptyline, paroxetine,        propantheline bromide, modafinil, fluoxetine, phenazopyridine,        methylprednisolone, carbamazepine, imipramine, diazepam,        sildenafil, bupropion, and sertraline.

Examples of combination therapies currently used are:

-   -   glatiramer acetate and albuterol,    -   glatiramer acetate and minocycline,    -   interferon-beta 1a and mycophenolate mofetil,    -   BHT-3009 and atorvastatin,

In a preferred embodiment of the present invention, the method fortreating multiple sclerosis in a subject in need thereof comprises theadministration to said subject of an effective amount of the medicamentor the pharmaceutical composition of the invention in combination withone or more therapeutic agent in the group of interferon-beta,glatiramer acetate, mitoxantrone, cyclophosphamide, methotrexate,aziathropine or natalizumab.

The present invention relates to the use of the pharmaceuticalcomposition or medicament of the invention, wherein the administrationto said subject of an effective amount of the medicament or thepharmaceutical composition of the invention is in combination with oneor more therapeutic agent in the group of interferon-beta, glatirameracetate, mitoxantrone, cyclophosphamide, methotrexate, aziathropine ornatalizumab.

In another embodiment, the present invention also relates to a method oftreatment of multiple sclerosis in which a the medicament or thepharmaceutical composition of the invention is to be administrated to asubject in need thereof, wherein the subject does not respond adequatelyto, or is unlikely to respond adequately to, one or more therapeuticagent in the group of interferon-beta, glatiramer acetate, mitoxantrone,cyclophosphamide, methotrexate, aziathropine or natalizumab.

The present invention relates to the use of the pharmaceuticalcomposition or medicament of the invention, wherein said subject doesnot respond adequately to, or is unlikely to respond adequately to, oneor more therapeutic agent in the group of interferon-beta, glatirameracetate, mitoxantrone, cyclophosphamide, methotrexate, aziathropine ornatalizumab.

“Inadequate response”, “does not respond adequately to”, or “unlikely torespond adequately” refer to an actual or probable response by a subjectwhich indicates that the therapy has been, or is likely to be,ineffective, toxic, or poorly tolerated insofar as the subject isconcerned.

Subjects that do not respond adequately or are unlikely to respondadequately to conventional treatment for multiple sclerosis such astreatment with one or more therapeutic agent in the group ofinterferon-beta, glatiramer acetate, mitoxantrone, cyclophosphamide,methotrexate, aziathropine or natalizumab, can be identified by usingthe EDSS score (Expanded Disability Status Scale) as conventionallyknown by the person skilled in the art.

Examples

In the following description, all experiments for which no detailedprotocol is given are performed according to standard protocol.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Experimental Procedures Mice

C57Bl/6 mice were obtained from Janvier (Le Genest-St-Isle, France).MOG35-55 specific-TCR transgenic mice on a C57Bl/6 background werehoused in the laboratory of Pr. Liblau (Inserm U563, Hopital Purpan,Toulouse). All mice were 7- to 8-weeks old females.

Antibodies and Reagents

The following antibodies were used for mouse cell purification andcharacterization: anti-CD4 (H129-19) anti-CD62L (Mel-14),(BD-Pharmingen, Le Pont de Claix, France). MOG₃₅₋₅₅ peptide was fromBachem (Voisin-le-Bretonneux, France). IL-2 was obtained by ChironCorporation (Emmeryville, Calif., USA). IL-4, IL-12 and anti-IL12 werepurchased from R&D systems (Minneapolis, USA).

T-Cells Purification and Culture

The medium used for T cell cultures was Iscove medium (Invitrogen)supplemented with FCS, Yssel medium and β2-Mercaptoethanol (Sigma).Splenocytes from MOG₃₅₋₅₅-specific TCR transgenic mouse were firstlabeled with FITC-conjugated anti-CD62L and PE-conjugated anti-CD4.Then, CD4⁺CD62L⁺ T cells were sorted on a FACStar SE (Becton Dickinson,France). All populations were >98% pure on reanalysis. The mouse Th1,Th2 and Tr1 cell directed against MOG₃₅₋₅₅ were obtained after in vitrodifferentiation as followed: 2.5×10⁵ Sorted CD4⁺CD62L⁺ T cells werecultured in the presence of 4.10⁶ irradiated syngeneic splenocytes in24-well plates and in presence of the MOG₃₅₋₅₅ peptide (10 μg/ml). IL-12(20 ng/ml), IL-4 (40 ng/ml) plus anti-IL-12 (5 μg/ml) or IL-10 (50ng/ml) was added for T cell differentiation into Th1, Th2 and Tr1 cells,respectively. Cells were cultured at 37° C., 5% CO2 and divided whenrequired in medium supplemented with IL-2 (100 UI/ml) for Th1 and IL-2plus IL-4 (20 ng/ml) for Th2 and Tr1 cells. Alternatively, Tr1 cellswere also divided in medium supplemented with IL-10 (5 ng/ml). T-cellpopulations were restimulated once a week during two or three weeks.

Cytokine Assays

Sandwich ELISAs were performed on 48 hours supernatants of anti-CD3 (10μg/ml)+anti-CD28 (1 μg/ml) stimulated T-cell populations. Briefly, 5.10⁵cells were activated with coated anti-CD3 and soluble anti-CD28monoclonal antibodies in 96-well flat bottom plates and cultured 48hours at 37° C., 5% CO₂. ELISAs were performed using anti-IL-4 (11B11),anti-IL-10 (2A5), anti-IFN-γ (XGM1.2), biotin anti-IL-4 (24G2),anti-IL-10 (SXC1), anti-IFN-γ (R4-6A2) (Pharmingen Becton Dickinson).

Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis was performed following theprotocol described by Cua et al (J. Exp. Med, 1999). Briefly, C57BL/6mice were injected intradermaly with 2.5 mg of Mouse spinal CordHomogenates prepared in Complete Freund Adjuvent. After 2 days, micewere injected with 200 ng of Pertussis Toxin by intraperitonealadministration. The same regimen of immunization was repeated at J8 andJ9 for Spinal Cord and Pertussis Toxin, respectively. Clinical scoreswere evaluated on a daily basis from Day 10 and are: 0=No disease;1=Tail paralysis; 2=Hind limb weakness; 3=Hind limb paralysis; 4=Hindlimb plus forelimb paralysis; 5=Moribund. T cell populations (3.10⁵cells/mouse) were injected once by intravenous route at day 9.

Results Differentiation of Anti-MOG₃₅₋₅₅ T Lymphocytes

We first differentiate anti-MOG₃₅₋₅₅ CD4+ T lymphocytes populations fromnaive CD4+ T lymphocytes isolated from anti-MOG₃₅₋₅₅ TCR transgenicmice. All lymphocytes from these mice bear a specific TCR thatspecifically recognize the peptide MOG₃₅₋₅₅ presented in the context ofH-2^(b) molecules. Naive cells (CD4⁺CD62L⁺) were sorted and activated invitro with irradiated syngeneic splenocytes and the MOG₃₅₋₅₅ peptide.IL-12 was added to differentiate Th1 cells, IL-4 and anti-IL12monoclonal antibodies were added to differentiate Th2 cells and IL-10was added to differentiate Tr1 cells. After 2 or 3 weekly stimulations,cells were harvested and tested for their production of cytokines underanti-CD3+anti-CD28 stimulation. Results are shown FIG. 1.

We observed that cells submitted to differentiation in the presence ofIL-10 acquired the typical pattern of Tr1 cell cytokine production withhigh IL-10 and low IL-4 production. IL-4 stimulation of differentiatingcells gave rise to Th2 cells showing equal production of IL-4 and IL-10and no IFN-γ production. Despite a significant IL-10 production, IL-12stimulation of differentiating cells gave rise to cells showing Th1cytokine production profile with high production of IFN-γ and no IL-4production.

In Vivo Suppressive Function of Anti-MOG₃₅₋₅₅ Tr1 Cells

We next evaluated the effect of differentiated anti-MOG T cellpopulations on Experimental Autoimmune Encephalomyelitis in mice. Forthis purpose, C57Bl/6 mice were immunized with mouse spinal cordhomogenates (msch) in complete freund adjuvant followed one day after byan intraperitoneal administration of Pertussis Toxin. The same regimenwas repeated at day 8 and 9 for msch and pertussis toxin, respectively.Anti-MOG Th1, Th2 and Tr1 cell populations were injected intravenouslyto immunized mice at day 9 and the clinical score was evaluated once aday. FIG. 2 shows the impact of anti-MOG CD4 positive T cells on theevolution of experimental encephalomyelitis. We observed that Th1 andTh2 cells directed against the MOG₃₅₋₅₅ peptide had no significanteffects on EAE induced by msch immunization. In contrast, administrationof anti-MOG₃₅₋₅₅ Tr1 cells dramatically inhibits the development of EAEin immunized mice. Indeed, mice treated with Tr1 cells directed againstmyelin antigen developed a mild tail paralysis whereas control micedeveloped a loss of motor function of the hind limbs. Importantly, Tr1administration not only inhibits the development of the illness but alsoprevent the relapse that occurs in non-T cell treated animals. Aprevious study (Barrat et al., J Exp Med, 2002) showed thatanti-ovalbumin Tr1 cells are able to prevent EAE in mice. Thesesuppressive effects were only achieved after intracranial instillationof ovalbumin showing that specific antigen activation of Tr1 cells inthe brain is a prerequisite for their effector function. We thus wantedto evaluate whether a self-antigen, as a intrinsic component of thecentral nervous system could play such a role of suppressor cellactivator. Our experiments answer positively to that question showingthat Tr1 cells directed against a myelin antigen can inhibitencephalomyelitis in vivo. The MOG protein is one of the targets ofpro-inflammatory cells in this mouse model of brain inflammation. Thefact that Tr1 cells directed against the same antigen suppressesinflammation suggests that Tr1 treatment of inflammatory diseases coulddirectly target the antigen for which tolerance was broken.

1. A pharmaceutical composition comprising at least one Tr1 cellpopulation directed against a multiple sclerosis-associated antigen incombination with one or more pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition according to claim 1, wherein said Tr1 cellpopulation is a Tr1 clone population.
 3. The pharmaceutical compositionaccording to claim 1, wherein said multiple sclerosis-associated antigenis selected from the group consisting of myelin basic protein, myelinassociated glycoprotein, myelin oligodendrocyte protein, proteolipidprotein, oligodendrocyte myelin oligoprotein, myelin associatedoligodendrocyte basic protein, oligodendrocyte specific protein, heatshock proteins, oligodendrocyte specific proteins, NOGO A, glycoproteinPo, peripheral myelin protein 22, 2′3′-cyclic nucleotide3′-phosphodiesterase, and fragments, variants and mixtures thereof. 4.The pharmaceutical composition according to claim 1, wherein saidmultiple sclerosis-associated antigen is selected from the groupconsisting of myelin basic protein (MBP), myelin associated glycoprotein(MAG), proteolipid protein (PLP), myelin oligodendrocyte protein (MOG)peptides, and fragments, variants and mixtures thereof.
 5. Thepharmaceutical composition according to claim 1, wherein said multiplesclerosis-associated antigen is selected from the group consisting ofmyelin basic protein (MBP), proteolipid protein (PLP) and myelinoligodendrocyte protein (MOG) peptides, and fragments, variants andmixtures thereof.
 6. The pharmaceutical composition according to claim1, wherein said multiple sclerosis-associated antigen is selected fromthe group consisting of peptides MBP 82-98, MBP 83-99, MBP 151-170, MBP111-129, and MBP 116-123.
 7. The pharmaceutical composition according toclaim 1, wherein said multiple sclerosis-associated antigen is selectedfrom the group consisting of peptides MOG 35-55, MOG 21-40, MOG 41-60,MOG 71-90, MOG 81-100, MOG 111-130, MOG 63-37, MOG 97-108, and MOG181-200.
 8. The pharmaceutical composition according to claim 1, whereinsaid pharmaceutical composition is frozen.
 9. The pharmaceuticalcomposition according to claim 1, wherein said pharmaceuticalcomposition further comprises one or more therapeutic agent that may beused for treating multiple sclerosis.
 10. The pharmaceutical compositionaccording to claim 1, wherein said pharmaceutical composition furthercomprises one or more therapeutic agent selected from the groupconsisting of interferon-beta, glatiramer acetate, mitoxantrone,cyclophosphamide, methotrexate, aziathropine, and natalizumab.